Presently practiced methods for the separation and quantification of oligonucleotide samples have several significant limitations. First, oligonucleotides are polyanionic species. In almost all HPLC-MS applications, e.g., electrospray ionization (ESI) systems employing hexafluoroisopropyl alcohol, the signal due to the oligonucleotide is divided among several different charge states. This males detection and quantitation, especially of impurities that are present at low levels, challenging because the signal-to-noise for each individual charge state is reduced. If one chooses to quantitate over multiple charge states, the method quickly becomes complicated and difficult to use for routine quality control (QC) use.
A second important consideration when using mass spectrometry for quantitative oligonucleotide analysis is that molecules of different lengths can have very different ionization efficiencies. Shorter oligonucleotides often ionize with much greater efficiency than longer oligonucleotides. This makes quantitation of a mixture of oligonucleotides of varying lengths by mass spectrometry difficult because one must determine the relative ionization efficiencies beforehand.
Thirdly, unlike the UV response of oligonucleotides, which obeys Beer's law at analytically relevant concentrations, the mass spectral response in ESI-MS across the same concentration range is not linear. This is believed to be due to ion suppression. In essence, at high oligonucleotide concentrations there is insufficient space on a single electrospray droplet to accommodate in a linear fashion more and more molecules for ionization. This results in a plateauing of the response at higher concentrations.
There is a need in the art for improved methods of separation and quantification of oligonucleotides.